RI基因siRNA干扰质粒的构建及其对人膀胱癌细胞迁移和侵袭能力的影响

RI Silencing by Small Interfering RNA Promotes Metastasis and Invasion of Human Bladder Cancer Cells

目的构建核糖核酸酶抑制因子(RI)基因siRNA干扰质粒,并检测其对人膀胱癌细胞BIU-87迁移和侵袭能力的影响。方法设计并构建针对RI基因mRNA的2个特异性siRNA表达载体和1个无同源性的阴性对照载体,经酶切和DNA测序鉴定后,转染BIU-87细胞,通过RT-PCR、Western blot和免疫细胞化学法检测干扰的有效性。光镜及HE染色观察细胞形态的改变,黏附试验、划痕试验和Transwell法检测细胞黏附、迁移和侵袭能力的变化,激光共聚焦显微镜观察细胞骨架微丝结构。结果酶切和测序结果证明干扰质粒构建正确;转染特异性siRNA表达载体的BIU-87细胞RI蛋白的表达水平明显降低,对照组未发生明显改变;转染干扰质粒的细胞堆叠严重,细胞黏附能力显著降低,迁移和侵袭能力显著提高,细胞骨架微丝聚集,伸展出片状伪足。结论已成功构建RI基因siRNA干扰质粒,其可显著提高膀胱癌细胞的迁移和侵袭能力,说明RI可作为抑制肿瘤迁移和侵袭的有效靶点。

Objective To construct the siRNA expression vectors and investigate the effect of RI expression silencing caused by vector-mediated RNAi on the metastasis and invasion of human bladder cancer BIU-87 cells. Methods Two siRNA expression vectors specific to RI mRNA and one non-homologous negative control vector were designed and constructed, then identified by restriction analysis and DNA sequencing and transfected to BIU-87 ceils. The efficiency of RNAi was determined by RT-PCR, Western blot and immunocytochemical method. The cells were observed for morphological change by light microscopy and HE staining, and determined for adhesion ability by adhesion test, for metastasis ability by wound healing assay, and for invasion ability by Transwell invasion assay. The microfilament structure of cytoskeleton was observed by laser scanning confocal microscopy. Results Both restriction analysis and sequencing proved that the siRNA expression vectors were constructed correctly. The expression level of RI protein in BIU-87 cells transfected with specific siRNA expression vectors decreased significantly, while that with negative control vector showed no significant change. The cells transfected with specific siRNA expression vectors overlapped severely, and their adhesion ability decreased significantly, while metastasis and invasion abilities increased significantly. Assembled cytoskeleton microfilament and several extended lamellipodia were observed in cells transfected with specific siRNA expression vectors. Conclusion The siRNA expression vectors specific to RI gene was successfully constructed, which increased the metastasis and invasion abilities of bladder cancer cells, indicating that RI might be used as an effective target for inhibitinz the metastasis and invasion of tumors.